Regulatory mechanisms of exopolysaccharide synthesis and biofilm formation in Streptococcus mutans

ABSTRACT Background Dental caries is a chronic, multifactorial and biofilm-mediated oral bacterial infection affecting almost every age group and every geographical region. Streptococcus mutans is considered an important pathogen responsible for the initiation and development of dental caries. It produces exopolysaccharides in situ to promote the colonization of cariogenic bacteria and coordinate dental biofilm development. Objective The understanding of the regulatory mechanism of S. mutans biofilm formation can provide a theoretical basis for the prevention and treatment of caries. Design At present, an increasing number of studies have identified many regulatory systems in S. mutans that regulate biofilm formation, including second messengers (e.g. c-di-AMP, Ap4A), transcription factors (e.g. EpsR, RcrR, StsR, AhrC, FruR), two-component systems (e.g. CovR, VicR), small RNA (including sRNA0426, srn92532, and srn133489), acetylation modifications (e.g. ActG), CRISPR-associated proteins (e.g. Cas3), PTS systems (e.g. EIIAB), quorum-sensing signaling system (e.g. LuxS), enzymes (including Dex, YidC, CopZ, EzrA, lmrB, SprV, RecA, PdxR, MurI) and small-molecule metabolites. Results This review summarizes the recent progress in the molecular regulatory mechanisms of exopolysaccharides synthesis and biofilm formation in S. mutans.


Introduction
Dental caries is a common chronic infectious disease caused by bacteria that seriously endangers the oral and systemic health [1,2]. According to the Global Burden of Disease (GBD) 2019 study, the caries rate of permanent teeth ranks first among 369 major diseases [3]. An estimated 2.0 billion (95% uncertainty interval, 1.8 to 2.3 billion) people with untreated caries of permanent teeth are present worldwide, and an age-standardized prevalence of 25,625.5 (22,281.1 to 29,372.0) per 100,000 person/year in 2019 [4,5]. Additionally, caries in deciduous teeth ranks first among all disorders in children aged 0 to 14 years in 2019, with 0.5 billion (0.4 to 1.6 billion) cases of caries in deciduous teeth and an age-standardized prevalence of 7,672.9 (6,113.1 to 9,339.6) [4,5]. S. mutans is closely related to the initiation and development of caries due to its aciduric and acidic properties, and ability to synthetize glucan to form biofilm [6]. The ability to synthesize large amounts of glucan to form biofilm contributes to the permanent colonization of tooth surfaces by S. mutans and the local development of the extracellular polymer matrix [7].
S. mutans biofilm formation is regulated by two independent mechanisms: sucrose-dependent and sucroseindependent [8,9]. In the absence of sucrose or early stages of adhesion, the interaction of antigen I/II (also known as P1, SpaP, Ag I/II, or PAc) with agglutinins in the saliva mediates the adhesion of S. mutans to the dental surfaces and bacterial aggregation [7,[10][11][12]. In the presence of sucrose, glucosyltransferases secreted by S. mutans, including GtfB, GtfC and GtfD, utilizes the glucose part of sucrose as a substrate to form the growing polymer of glucan, which is also called exopolysaccharides [7,13]. The presence of exopolysaccharides enhances the strength of the local adhesion of S. mutans to the tooth surfaces [14]. Moreover, the continuous production of exopolysaccharides in situ further expanded the three-dimensional matrix while forming a bacterial cell core wrapped by exopolysaccharides [15]. It also provides a scaffold for the colonization of other microorganisms (e.g. Candida albicans) and the adhesion of other substances (including eDNA, lipoteichoic acids, proteins), further leading to biofilm formation [16].
This review summarizes the molecular regulatory mechanisms affecting the formation of S. mutans biofilm. The signals that regulate the formation of S. mutans biofilm are considered from five different aspects: (1) upstream signals, such as second messengers; (2) transcriptional level, such as transcription factors and two-component systems; (3) posttranscriptional level, such as small RNAs; (4) posttranslational level, such as acetylation, malonylation; (5) others, such as CRISPR-related proteins, PTS systems, quorum-sensing signaling system, enzymes and small-molecule metabolites ( Figure 1).

Second messengers
Bacteria use second messengers to conduct signals in response to the rapid change of the environment. In recent years, many nucleotide molecules or second messengers have been found as regulators of various physiological activities of bacteria, including biofilm formation [17].

c-di-AMP
c-di-AMP is an important and ubiquitous second messenger in bacterial signaling, whose intracellular levels are regulated by the enzymes diadenylate cyclase (DAC) and phosphodiesterase (PDE) [18]. DAC contains a highly conserved DAC/DisA_N domain, which utilizes two molecules of ATP or ADP as a substrate to synthesize c-di-AMP [19,20]. PDE contains a DHH/DHHA1 (Asp-His-His)/HD (Asp-His) domain, which breaks down c-di-AMP into two AMP molecules or 5′phosphoadenylyl-(3′−5′)-adenosine (5'-pApA) [21,22]. Therefore, the DAC and PDE processes keep the intracellular c-di-AMP level within an appropriate range.
The DAC and PDE enzymes in S. mutans are encoded by cdaA and pdeA, respectively. Cheng et al. found that the c-di-AMP levels of S. mutans decreased significantly after knockout cdaA [23]. Genes with increased expression were clustered in cellular polysaccharide biosynthetic processes, including gtfB, gtfC [23]. Besides, both GTF enzyme activity and their expression at the gene level were significantly increased. Nevertheless, Peng et al. constructed a similar in-frame deletion mutant obtaining different results [24]. Indeed, DAC deficiency significantly reduces the expression of gtfB and inhibits the synthesis of exopolysaccharides, but does not markedly affect the expression of gtfC and gtfD. Therefore, the involvement of c-di-AMP in the formation of S. mutans biofilms occurs through the regulation of gtfB expression. In addition, Peng et al. revealed that CabpA and CabpB are two receptor proteins for c-di-AMP [25]. CabPA interacts with VicR, a response regulator that regulates Figure 1. The regulatory mechanisms of exopolysaccharides synthesis and biofilm formation in S. mutans. c-di-AMP controls S. mutans biofilm formation by regulating gtfB expression through the binding of CabPA to VicR, while Ap4A represses the expression of gtfs through some unknown pathways. VicR, EpsR and CovR regulate S. mutans exopolysaccharides synthesis and biofilm formation directly by binding to the promoters of gtfB and gtfC. Post-translational modifications are also involved in the regulation of Gtfs activities and biofilm formation in S. mutans. gtfB expression, thereby promoting biofilm formation. These results demonstrate that c-di-AMP is an upstream signal that regulates biofilm formation in S. mutans.

Ap4A
Ap4A is a dinucleotide metabolite that consists of two adenosines joined in the 5′−5′ linkage by four phosphates [26,27]. During translation, aminoacyl-tRNA synthetases (AARS) catalyze the transfer of the AMP moiety from the aminoacyl adenylate to the ATP to form Ap4A in the absence of tRNA [28]. Ap4A is symmetrically decomposed into two molecules of ADP or asymmetrically into AMP and ATP by the Ap4A hydrolase YqeK, which was identified and characterized in S. mutans [29]. Zheng et al. showed that the in-frame deletion of the yqeK gene in S. mutans causes an increase in the intracellular Ap4A levels, while biofilm formation and waterinsoluble exopolysaccharides production are decreased [30]. The knockout of the yqeK gene leads to the downregulation of important virulence genes related to biofilms, such as gtfB, gtfC, and gtfD [30]. Although the mechanisms involved in the role of Ap4A need to be further investigated, these results suggest that it affects biofilm formation in S. mutans by affecting the expression of gtfs and the activity of Gtfs.

Transcription factors
Some transcription factors also play a very important role in the formation of S. mutans biofilm. The exopolysaccharides synthesis regulator (epsR) gene is one of the transcription factors of the multiple antibiotic resistance regulator (MarR) family in S. mutans. Chen et al. demonstrate that EpsR specifically binds to the promoter regions of gtfB, consequently negatively regulating gtfB expression and exopolysaccharides production in S. mutans [31].
RcrR, a rel competence-related regulator, is a transcription factor belonging to the multiple antibiotic resistance regulator (MarR) family in S. mutans. RcrR specifically binds to the promoter regions of Smu.1185 and Smu.2038 to regulate the carbohydrate transport of mannitol and trehalosespecific PTS system, and it also has a certain impact on the transport of several carbohydrates, including glucose, galactose, lactose, and maltose [32]. The deletion of the rcrR gene in S. mutans decreases the ability to form the biofilm and affects the expression of its multiple genes related to sugar transportation [32].
StsR is the transcription factors of GntR family that specifically binds to the predicted promoter sequences in the mannitol-specific PTS transporter, the mannose-specific PTS transporter, maltose ABC transporter and multiple sugar-binding ABC transporter in S. mutans [33]. When stsR is knocked out, the amount of biofilm and the content of exopolysaccharides of S. mutans decreases significantly at the early stage [33].
AhrC is a transcription factor of the arginine repressor (ArgR) family in S. mutans. The results of electrophoretic mobility shift assay (EMSA) and DNase I footprinting showed that AhrC specifically binds to the argC promoter region [34]. AhrC also binds to the promoter regions of argG, Smu.815, Smu.1904, and Smu.940c to regulate the downstream gene expression [34]. In addition, the S. mutans strain overexpressing ahrC has a significant decrease in the formation of biofilm and content of water-insoluble exopolysaccharides [34]. These results suggest that AhrC negatively regulates arginine biosynthesis and biofilm formation in S. mutans.
FruR is a deoxyribonucleoside repressor (deoR)type regulator that co-transcribes a single EIIABC Fru permease with FruI, and a putative 1-phosphofructokinase (1-PFK) with FruK. The loss of FruR results in a significantly reduction of S. mutans biofilm [35].
PdxR is encoded by smu.864, a member of the GntR superfamily of regulatory proteins. When S. mutans is grown in defined medium, pdxR deficiency affects its growth of and significantly reduces biofilm formation [36].
A total of 130 transcription factors were predicted in S. mutans (https://mistdb.com/). However, the functions of several transcription factors remain unknown. Therefore, further research is necessary to investigate the relationships between the uncharacterized transcription factors and biofilm formation.

Two-component systems
Bacterial two-component systems are regulatory circuits of signal transduction based typically on a membrane bound sensor histidine kinase (HK) and a cytoplasmic response regulator (RR) that is activated through a histidine to aspartate phosphorelay reaction [37]. At present, 14 two-component systems and one orphan regulator (CovR) have been found in S. mutans [38]. Among them, VicR positively regulates the expression of gtfs by binding to the promoter region of gtfB and gtfC [39,40]. CovR (also known as GcrR) is a negative regulator that inhibits the expression of gtfB and gtfC by directly binding to the promoter region [41].
Zhang et al. constructed a series of mutant strains (including vicR+covR+, vicR and covR overexpression; vicR+cov-, vicR overexpression and covR deficiency; ASvicRcovR+, vicR low-expression and covR overexpression; ASvicRcovR−, vicR low-expression and covR deficient) to explore the synergy between VicR and CovR in S. mutans in the regulation of the sucrose-selective exopolysaccharides metabolism, and they found that gtfB/gtfC expression is mainly regulated by covR regardless of the vicR gene expression, as revealed by the analysis of the biofilm content of these several strains [42]. These results suggest that CovR may play a dominant role in the interaction between CovR and VicR affecting the expression of gtfs.
VicK is a histidine protein kinase that monitors and transmits chemical signals to the downstream regulatory proteins such as VicR and CovR in S. mutans [43]. Subsequently, VicR or CovR regulate biofilm-associated genes at a transcriptional level, such as gtfB/C, ftf and gbpB [44]. A study found that the deletion of the vicK gene in S. mutans suppresses biofilm formation as well as exopolysaccharides production, and the expression of genes related to exopolysaccharides synthesis are down-regulated, with the exception of gtfB [45]. In addition, the expression of vicX and covR genes are decreased after vicK knockout. These results indicate that VicK regulates biofilm formation by affecting CovR and VicR.

Regulation of S. mutans biofilm formation at the post-transcriptional level
Small RNAs (sRNAs) in bacteria are typically 50-400 nucleotides in length, playing a key role in regulating gene expression [46,47]. In the canonical pathway regulating sRNA-mediated gene expression, sRNAs, often along with the chaperone protein Hfq, positively or negatively regulate the target mRNA expression in response to environmental changes through an incomplete Watson-Crick base pairing [48]. Some studies discovered the presence of many sRNAs with regulatory functions in S. mutans [49][50][51]. Yin et al. detected a potential sRNA regulation pathway in S. mutans by bioinformatics technology [52]. The results showed that sRNA0426 has a strong positive relationship with the dynamic biofilm formation in S. mutans. Furthermore, the expression of gtfB and gtfC mRNAs are positively correlated with sRNA0426 expression. Liu et al. evaluated the expression of sRNAs and target genes (gtfB, gtfC, and spaP) related to virulence, and they found that the target mRNA of srn92530 is gtfB, while the target mRNA of srn92532 and srn133489 is gtfB and gtfC, respectively [53].
The rnc gene is considered the coding gene of ribonuclease III (RNase III), which can promote mRNA maturation [54]. Mao et al. observed that the rnc gene is located upstream of the vicRKX tricistronic operon and the downstream vic locus is repressed by rnc at the mRNA level in S. mutans. Three putative microRNA-size small RNAs (msRNAs), such as msRNA1701, msRNA3405, and msRNA1657, are negatively correlated with vicRKX but positively correlated with rnc, as revealed by deep sequencing and bioinformatics analysis [55]. Moreover, they found that the knockout rnc gene reduces the ability of biofilm formation of S. mutans [56].
Antisense RNA (AS RNA) refers to an RNA that inhibits the expression of related genes after complementing with mRNA [57]. Lei et al. detected an AS RNA named ASvicR upstream of the rnc gene in S. mutans, whose overexpression inhibits the transcription of vicR, gtfB, gtfC and gtfD, resulting in the decrease of the exopolysaccharides synthesis and biofilm formation [58,59]. Moreover, msRNA1657, a microRNA-size small RNA, binds to the 5′-UTR region of the vicR gene [59,60]. This suggests that ASvicR and msRNA1657 regulate gtfs transcription through the vicR gene. At present, the detailed mechanisms of small RNA affecting the biofilm formation of S. mutans is still not well-known.

Regulation of S. mutans biofilm formation at the post-translation level
Protein translational modifications (PTMs) increase the functional diversity of proteome through the covalent addition of functional groups or proteins, the proteolytic cleavage of regulatory subunits or the degradation of the whole protein [61,62]. PTM has important functions in regulating the physiological activities of S. mutans. For example, glutathionylation on the Cys41 residue of Tlp is crucial to protect S. mutans from oxidative stress and in the competition with S. sanguinis and S. gordonii [63]. Lei et al. identified 973 acetylation sites in 445 proteins of S. mutans, and among them, 617 acetylation sites in 302 proteins were quantified, revealing that the activity of GtfB, GtfC, and GtfD is regulated by acetylation [64]. A recent study found that the GCN5-related N-acetyltransferases (GNAT) family member ActG catalyzes the acetylation of GtfB and GtfC in S. mutans, subsequently inhibiting the synthesis of water-insoluble exopolysaccharides and biofilm formation [65].
Li et al. performed a global protein lysine malonylation (Kmal) analysis of S. mutans and identified a total of 392 malonyllysine sites in 159 proteins [66]. Among them, proteins (WapA, SpaP, GtfC, Ftf, GbpB) closely related to S. mutans biofilm formation were modified with Kmal, suggesting that Kmal is related to exopolysaccharides synthesis and biofilm formation [66].

CRISPR-associated proteins
CRISPR clusters are DNA repeats family widely present in the genome of bacteria and archaea [67].
A CRISPR locus consists of a CRISPR array bordered by various cas genes, which are composed of short direct repeats separated by short variable DNA sequences (called spacers) [68,69]. Two CRISPR-Cas systems exist in S. mutans: the CRISPR1 system (type II-A) and the CRISPR2 system (type I-C) [70].
A study found that the expression of two CRISPR/ Cas locus sites in S. mutans is differentially regulated in cdaA knockout strains, which show an abnormal biofilm phenotype [23]. Zhang et al. found that S. mutans clinical strains with both CRIPSR systems have a stronger ability of biofilm formation [70]. Later, Tang et al. observed that the knockout of cas3 reduces the ability of S. mutans to produce exopolysaccharides and form biofilm by decreasing the expression of gtfB and gtfC [71]. This result suggests that the CRISPR-Cas systems are involved in the regulation of gtfs expression and biofilm formation in S. mutans.

PTS system
The PTS system consists of the nonspecific enzyme I (EI), histidine-containing the phosphocarrier protein (HPr), and sugar-specific enzyme II (EII) complexes. The EI and HPr are involved in the transport of all PTS sugars: HPr encodes ptsH and EI encodes ptsI in S. mutans [72]. The EII complex usually consists of three parts: EIIA, EIIB and EIIC, where EIIA and EIIB are located in the cytoplasm and EIIC is an integral membrane protein (mannose-type PTS system containing EIID) [73].
EII Man is the most physiologically important PTS complex in S. mutans, mainly involved in the transport of glucose, mannose, galactose, glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) [74,75]. This complex is made up of four domains expressed as three polypeptides in a single operon: EIIAB (manL), EIIC (manM), and EIID (manN). Previous studies showed that the deletion of the manL gene relieved CCR of several carbohydrate catabolic operons, including the cel operon encoding a phospho-βglucosidase (CelA), a cellobiose-PTS EII complex [76,77], the lac operon encoding EII Lac (lacFE) and proteins required for the utilization of both lactose and galactose [74]. Abranches et al. found that the reduced expression of the gtfBC promoter in the EIIAB Man mutant strain is associated to the involvement of ManL in the regulation of gtfBC expression [78].

Quorum-sensing signaling system
Quorum sensing is a mechanism of bacterial intercellular communication for the regulation of gene expression in response to the density of the population. It is based on the production, detection and response of extracellular signaling molecules known as autoinducers [79]. The LuxS system mediates the interspecific interactions in multi-species communities, and it is one of the widely studied quorumsensing systems in S. mutans [38].
The luxS gene is involved in many cellular processes, and it is present in a wide range of bacteria [80]. LuxS catalyzes the formation of the autoinducer AI−2 (a furanosyl borate diester) in the methyl cycle [81]. A study found that the knockout of luxS in S. mutans suppresses gtfD expression [82]. In addition, its knockdown affects S. mutans ABC transporters and carbohydrate transport, transformation, and metabolism through the EII subunits and enzymes to influence virulence-associated traits, as revealed by a transcriptome analysis [81]. Thus, the quorumsensing signaling system in S. mutans is involved in the formation of biofilms.

Enzymes
Dextranase (Dex) is a glucanase involved in the degradation of water-soluble glucans, and includes DexA and DexB [83]. DexA breaks the α−1,6-linkage of the extracellular glucans to produce oligosaccharides, which are degraded into monosaccharides by DexB glucosidase after entering the cells [84][85][86]. Yang et al. revealed that dexA knockout results in an increased transcription of genes related to exopolysaccharides synthesis, including gtfB, gtfD and ftf [87]. In addition, the biofilms of the strains overexpressing dexA lack exopolysaccharides matrix and are unable to aggregate into dense and continuous microcolonies [87]. These results indicate an important role of the dexA gene in the formation of S. mutans biofilm.
YidC proteins are membrane-localized chaperone insertases that are universally conserved in all bacteria [88]. The genome of S. mutans contains two copies of genes encoding the YidC homologs yidC1 and yidC2, and their protein sequences are 27% identical and 48% similar. The deletion of yidC1 or yidC2 results in a reduced synthesis of insoluble glucan at the early and mid-exponential growth phases [89]. In particular, the deletion of yidC2 resulted in a significant reduction in biofilm biomass, evident defects in the spatial organization of the extracellular polymer matrix, and alteration in the threedimensional biofilm structure [89]. The defective biofilm contains smaller bacterial clusters with higher cell density and less surrounding exopolysaccharides than the biofilm in the wild type [89].
The copYAZ is a copper-transport operon in S. mutans that encodes three proteins: a copper P-type ATPases (CopA), a copper-responsive repressor (CopY), and a copper chaperone (CopZ) [90]. CopY allows transcription upon copper transfer from direct binding with CopZ [90,91]. CopA is a small protein that tightly binds to copper and delivers it to CopY to positively regulate copYAZ [90][91][92]. CopZ exports the excess copper ions to the outside of the cell, causing the cell to become resistant to copper [90,91]. Garcia et al. found that glycosyltransferase secretion and biofilm formation are significantly reduced after copZ knockdown in S. mutans [93]. This result suggests that CopZ is a global regulator of biofilms. Further characterization of CopZ may lead to the identification of novel pathways in biofilm formation and characterization.
EzrA is an integral membrane protein consisting of an N-terminal trans-membrane (TM) spanning helix followed by an 60 kDa cytoplasmic domain [94]. It plays a key role in the regulation of cell division and in the maintenance of cell size and shape [95][96][97]. Xiang et al. investigated the role of EzrA in S. mutans by constructing ezrA in-frame deletion mutants. The results showed that the ezrA mutant grows slower that the wild type, with a round cell shape characterized by a shortened length and extended width [98]. Single-species cariogenic biofilm model revealed that the deletion of ezrA results in a defective biofilm formation with less exopolysaccharides and altered three-dimensional biofilm architecture [98].
Amyloid is a fibrous cross-β sheet quaternary structure consisting of ordered peptides or polymerized proteins [99]. S. mutans is an amyloid-forming organism and amyloidogenesis contributes to biofilm formation in this bacterium [100]. Three proteins are present in S. mutans that form amyloidogenic fibrils, including amyloidogenic adhesin SpaP, wallassociated protein A (WapA) and the secreted protein Smu.63c [101]. When S. mutans grows in biofilm medium containing glucose as the sole carbon source, the strains with spaP and wapA deletion produce significantly less biofilm than the wild type strain, while the deletion of Smu.63c results in a slight increase in biofilm production [101]. The reduction of biofilm is more significant in the ΔspaP/ΔwapA double-deletion strain and the ΔspaP/Δsmu.63c/ ΔwapA triple-deletion strain [101]. ΔSmu.63c/ ΔwapA double-deletion strain and ΔSmu.63c/ΔspaP double-deletion strain rescued the spaP and wapA deletion phenotypes, resulting in biofilm production comparable to that of the wild-type strain [101]. These results suggest that SpaP and WapA positively regulate the biofilm formation in S. mutans, while Smu.63c exerts a negative regulatory role.
The efflux pump is a protein localized on the cell membrane that allows the maintenance of the homeostasis of the internal environment in the microorganisms by excreting toxic substances, such as antimicrobial drugs, metabolites and signal molecules of the quorum sensing [102]. LmrB is a putative efflux pump in S. mutans, and its inactivation results in the structural changes of the biofilm, increased production of exopolysaccharides and increased transcription of exopolysaccharides-related genes [103]. A number of genes involved in sugar uptake and metabolism are up-regulated, including sugar metabolism associated glg operons and msmREFGK transporter, as demonstrated by the transcriptome analysis [104].
The smu.833 gene encodes a hypothetical glycosyltransferase highly conserved among the group of mutans streptococci but absent in other oral commensals [105]. The protein encoded by smu.833 has two transmembrane helices and is highly homologous to proteins involved in cell wall biogenesis and the response to cellular stress [105]. Rainey et al. found that smu.833 regulates the dynamic interaction between glucan and extracellular DNA (eDNA) of S. mutans [105]. Moreover, the loss of smu.833 resulted in a significant decrease in the expression of Gtfs and a concurrent reduction in glucan matrix [105].
The streptococcal pleiotropic regulator of virulence (SprV) is a small hypothetical protein that is encoded by smu.2137 in S. mutans. The deletion of SprV leads to an impaired biofilm formation and various virulence-related functions in S. mutans [106]. Transcriptome sequencing revealed that gtfB, gbpA, gbpB, and gbpC, which are genes related to biofilm formation, are downregulated by 2.6-, 2.4-, 1.7-, and 2.6-fold, respectively [106].
Recombinase A (RecA) is related to the SOSresponse in S. mutans. The RecA-deficient mutant strain possesses a lower acid tolerance and produces a biofilm with a lower density than the wild type [107]. Glutamate racemase (MurI) is an essential enzyme for the biosynthesis of peptidoglycan. murI deficiency in S. mutans weakens the ability to form the biofilm and virulence factors. The expression of comE, comD, gtfB and gtfC, genes related to biofilm formation are down-regulated 8-, 43-, 85-and 298fold, as revealed by qRT-PCR [108].

Small-molecule metabolites
Small molecules are often encoded by biosynthetic gene clusters (BGCs) and are the primary means of communication in the microbial world. They are involved in various microbial-microbial and hostmicrobial interactions, including antimicrobial activity, bacterial signaling, immunomodulation, biofilm formation, host colonization, nutrient clearance, and stress protection [109]. The genomic analysis of S. mutans isolates revealed that it collectively harbors a plethora of BGCs for polyketide/non-ribosomal peptide biosynthesis [110].

The strategies for biofilm inhibition and caries prevention
Although caries is a multi-microbial disease, selective targeting of S. mutans has been considered to be an appropriate approach for the prevention of dental caries [112]. This is mainly because the synthesis of exopolysaccharides from sucrose by S. mutans is important to the development of cariogenic biofilms. Thus, targeted inhibition of S. mutans biofilm may be a viable approach to impede the progression of caries without disrupting the oral microbiome associated with health.
Gtfs secreted by S. mutans can synthesize exopolysaccharides using sucrose [113,114]. Therefore, targeting Gtfs could selectively prevent the synthesis of exopolysaccharides, the formation of biofilm and impair the S. mutans virulence, which do not threaten the microecological balance in the oral cavity. Screening for compounds that inhibit Gtfs could have a significant effect on biofilm inhibition. Fluoride can inhibit the production and secretion of S. mutans Gtfs [115]. Some sucrose structural analogs (including 6-deoxysucrose, 6-thiosucrose, 4,6-dideoxysucrose, sucralose, 4-deoxysucrose, 4-chloro−4-deoxygalactosucrose, 6,6'-dithiodisucrose) Smu.337 Deletion of yidC1 resulted in less insoluble glucan synthesis but produced more soluble glucans, especially at early and mid-exponential-growth phases. [89] yidC2 Smu.1727 Deletion of yidC2 resulted in a significant reduction in biofilm biomass and pronounced defects in the spatial organization of the extracellular polymer matrix.
Because the specificity of these compounds is largely unknown, their access to clinical applications remains limited. The upstream regulatory genes, including TCS and quorum sensing system, regulate S. mutans biofilm formation by affecting the promoter activity of gtfs and subsequent expression of Gtfs. Therefore, these upstream regulatory systems may be potential targets, which can be used to develop therapeutic agents against dental caries. A number of inhibitors targeting upstream regulatory pathways have been identified. WalKHK, a TCS inhibitor from B. subtilis, inhibits the in vitro autophosphorylation of VicK in S. mutans [124]. The cell extract from Tenacibaculum sp. 20J interfered specifically with the AI−2 quorum sensing system and reduced biofilm formation of S. mutans [125]. Although the expression of gtfs is well regulated, the exact mechanism of regulation is still not fully understood. If these regulatory genes also affect the expression of other genes (e.g. mutacins), then the strategies of targeting upstream regulatory genes may affect the oral microecological homeostasis.
Second messenger c-di-AMP and Ap4A regulate S. mutans biofilms formation. Inhibitors targeting synthetase/hydrolase or receptor proteins can affect the intracellular levels or signaling transduction of second messengers and inhibit biofilm formation. Currently, most c-di-AMP inhibitors target PDE and DisA [126]. For example, three inhibitors of Bacillus subtilis DisA, bromophenol-TH, suramin, and the tea polyphenol theaflavin digallate, were identified [126]. Stress alarmone ppGpp could competitively inhibit B. subtilis GdpP (PDE) [127]. However, inhibitors targeting S. mutans DAC/PDE still need further investigation. The mechanisms of action of these inhibitors and whether there might be an impact on the other flora in the oral cavity are still unclear.

Conclusions
S. mutans is the core microorganism causing human dental caries due to its high ability of biofilm formation and cariogenicity. The regulatory network of biofilm formation mainly includes upstream signals composed of c-di-AMP and Ap4A, regulating gtfs gene expression by CRISPR-Cas, transcription factor, two-component systems, small RNA, PTS systems, and modulating glucosyltransferases activity by acetylation, malonylation. The specific inhibition of S. mutans biofilm modulates the oral microecological balance. Therefore, targeting the systems that regulate the biofilm formation might provide potential strategies for the ecological prevention and treatment of caries.